An electron microscopic study of the ultrastructure of microbial cells in extreme biotopes in situ

The ultrastructure of microbial cells was studied in situ in natural biotopes by high-resolution transmission electron microscopy using the known methods of cryofractography, thin sectioning, and the negative staining of total cell specimens, as well as new methods of the low-temperature fractionation of microbial cells (providing for the recovery of cells from natural sources and their concentration), the preparation of micromonoliths, and aimed electron microscopy. Among the natural biotopes studied were permafrost ground and oil sludge. Most of the microorganisms found in the 1- to 3-million-year-old permafrost ground represented resting forms (spores, cysts, and cyst-like cells with specific organo-mineral envelopes). Oil sludge older than 35 years contained bacteria of atypical morphology and ultrastructure, including various resting forms and ultramicrobacteria. The data obtained is indicative of considerable promise of high-resolution electron microscopy in studying microbial communities in situ.     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1–3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29–43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of ≤ 0.3 μm and volume of ≤ 0.014 μm³) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18–0.20 × 0.20–0.30 μm in size, forming in situ spherical aggregates (microcolonies) of dividing cells. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Electron microscopic detection and in situ characterization of bacterial nanoforms in extreme biotopes

The morphology, ultrastructure, and quantity of bacterial nanoforms were studied in extreme biotopes: East Siberia permafrost soil (1-3 Ma old), petroleum-containing slimes (35 years old), and biofilms from subsurface oil pipelines. The morphology and ultrastructure of microbial cells in natural biotopes in situ were investigated by high-resolution transmission electron microscopy and various methods of sample preparation: ultrathin sectioning, cell replicas, and cryofractography. It was shown that the biotopes under study contained high numbers of bacterial nanoforms (29-43% of the total number of microorganisms) that could be assigned to ultramicrobacteria due to their size (diameter of < or =0.3 microm and volume of < or =0.014 microm3) and structural characteristics (the presence of the outer and cytoplasmic membranes, nucleoid, and cell wall, as well as their division patterns). Seven different morphostructural types of nanoforms of vegetative cells, as well as nanospores and cyst-like cells were described, potentially representing new species of ultramicrobacteria. In petroleum-containing slimes, a peculiar type of nanocells was discovered, gram-negative cells mostly 0.18-0.20 x 0.20-0.30 microm in size, forming spherical aggregates (microcolonies) of dividing cells in situ. The data obtained promoted the isolation of pure cultures of ultramicrobacteria from petroleum-containing slimes; they resembled the ultramicrobacterium observed in situ in their morphology and ultrastructure.     

Formation of resting forms of arthrobacter globiformis in autolyzing cell suspensions

Under conditions of the spontaneous or induced autolysis of thick cell suspensions,Arthrobacter globiformis strains produced cells exhibiting features typical of resting microbial forms. The number of viable resting cells was greater under conditions of induced rather than spontaneous autolysis. The thermoresistance of the resting cells of A.globiformis strains isolated from 2-to 3 million-year-old permafrost was higher than that of the collectionA. globiformis strain.     

Formation of resting forms of Arthrobacter globiformis in autolysing cell suspensions

Under conditions of spontaneous or induced autolysis of thick cell suspensions, Arthrobacter globiformis strains produced cells exhibiting features typical of resting microbial forms. The number of viable resting cells was greater under conditions of induced rather than spontaneous autolysis. The thermoresistance of the resting cells of A. globiformis strains isolated from 2- to 3 million-year-old permafrost was higher than that of the collection A. globiformis strain.     

Morphology and ultrastructure of the testes and spermatozoa of the South African leopard (Panthera pardus)

The survival of South African leopard (Panthera pardus) populations is at risk due to the decrease in natural habitat caused by increasing human populations, development projects and industry. The use of ex situ conservation methods such as assisted reproductive techniques (ARTs) in captive breeding programmes can be used to conserve and protect this species. However, a good understanding of the reproductive system of the species is imperative in the development of successful ARTs. This study serves to provide information on the morphology and ultrastructure of the spermatozoa and testes of P. pardus that can be used to help in the development, testing and optimization of reproductive techniques. The objectives of this study were (i) to assess the morphology and ultrastructure of the spermatozoa by means of fluorescence and electron microscopy respectively, and (ii) to examine the morphology and ultrastructure of the testicular tissue as well as of the stages of spermatogenesis using light and electron microscopy. In this study, it was noted that the morphology and ultrastructure of the spermatozoa are similar to those reported in other feline and mammalian species and that a high amount of morphologically abnormal ejaculated sperm was found to be a common occurrence in all samples obtained.     

Reactivation of dormant and nonculturable bacterial forms from paleosoils and subsoil permafrost

Methods of reactivating the dormant forms (DFs) and nonculturable cells (NCs) of the bacterial communities of buried paleosoils and subsoil permafrost stored for long periods of time (thousands to millions of years), including completely sterile samples (CFU = 0), were developed. They were based on washing the DFs and NCs to remove anabiosis autoinducers (spore germination autoinhibitors) and introducing low molecular weight extracellular growth regulators of microbial or plant origin, such as alkylhydroxybenzenes of the alkylresorcinol subtype, indoleacetic acid, and wheat germ agglutinin. It was revealed that the dormant communities of permafrost and buried soils differed in their sensitivity to reactivating factors, probably due to different natural storage conditions of the tested soil substrates and the heterogeneity of dormant populations. The latter was confirmed by FISH (fluorescent in situ hybridization): applying the reactivation methods to the cells of the dormant permafrost community resulted in an increase in the number of metabolically active cells from 5 to 77% of their total number. In contrast, the addition of microbial anabiosis autoinducers (C₁₂-AHB) to background surface soil and permafrost samples caused the transition of bacterial cells to the dormant or the nonculturable state, depending on the C₁₂-AHB concentration and the sensitivity of the cells from the control soil or permafrost’ to it. The data obtained contribute to our knowledge concerning the role of intercellular communication factors and the survival of microorganisms under extreme environmental conditions.     

Reproductive resting forms ofArthrobacter globiformis

Submerged cultures ofArthrobacter globiformis grown in media unbalanced with respect to carbon and nitrogen sources were found to contain cells exhibiting features typical of resting forms: long-term viability, specific ultrastructure, dormant metabolism, and thermoresistance. Such cells were produced not only in the collection strain VKM B-l 112, but also in the Aglobiformis strains isolated from 2-to 3-million-year-old permafrost sediments.     

Reproductive resting forms of Arthrobacter globiformis

Submerged cultures of Arthrobacter globiformis grown in media unbalanced with respect to carbon and nitrogen sources were found to contain cells exhibiting features typical of resting forms: long-term viability, specific ultrastructure, dormant metabolism, and thermoresistance. Such cells were produced not only in the collection strain VKM B-1112, but also in the A. globiformis strains isolated from 2- to 3-million-year-old permafrost sediments.     

Microscopic examination of bacteria in Fe(III)-oxide deposited from ground water

Ochreous sludge deposited in the course of aeration of ground water contained an assortment of bacterial forms and structures which were investigated by light microscopy, scanning electron microscopy, and transmission electron microscopy. Bacterial structures were often covered by iron deposition which could be removed by acidification of the samples. Sulfuric acid treatment was consistently better than hydrochloric acid to dissolve iron without a considerable damage to the bacterial cells. Partial dissolution of amorphous ferric iron was achieved by acidifying the samples with oxalic acid or citric acid prior to the preparation for electron microscopy.     

Ultrastructure of Resting Cells of Some Non-Spore-Forming Bacteria

Using electron microscopy (ultrathin sections and freeze-fractures), we investigated the ultrastructure of the resting cells formed in cultures of Micrococcus luteus, Arthrobacter globiformis, and Pseudomonas aurantiaca under conditions of prolonged incubation (up to 9 months). These resting cells included cystlike forms that were characterized by a complex cell structure and the following ultrastructural properties: (i) a thickened or multiprofiled cell wall (CW), typically made up of a layer of the preexisting CW and one to three de novo synthesized murein layers; (ii) a thick, structurally differentiated capsule; (iii) the presence of large intramembrane particles (d = 180–270 Å), occurring both on the PF and EF faces of the membrane fractures of M. luteus and A. globiformis; (iv) a peculiar structure of the cytoplasm, which was either fine-grained or lumpy (coarse-grained) in different parts of the cell population; and (v) a condensed nucleoid. Intense formation of cystlike cells occurred in aged (2- to 9-month-old) bacterial cultures grown on diluted complex media or on nitrogen-, carbon-, and phosphorus-limited synthetic media, as well as in cell suspensions incubated in media with sodium silicate. The general morphological properties, ultrastructural organization, and physiological features of cystlike cells formed during the developmental cycle suggest that constitutive dormancy is characteristic of non-spore-forming bacteria.     

Cluster Structure of Anaerobic Aggregates of an Expanded Granular Sludge Bed Reactor

The metabolic properties and ultrastructure of mesophilic aggregates from a full-scale expanded granular sludge bed reactor treating brewery wastewater are described. The aggregates had a very high methanogenic activity on acetate (17.19 mmol of CH₄/g of volatile suspended solids [VSS]·day or 1.1 g of CH₄ chemical oxygen demand/g of VSS·day). Fluorescent in situ hybridization using 16S rRNA probes of crushed granules showed that 70 and 30% of the cells belonged to the archaebacterial and eubacterial domains, respectively. The spherical aggregates were black but contained numerous whitish spots on their surfaces. Cross-sectioning these aggregates revealed that the white spots appeared to be white clusters embedded in a black matrix. The white clusters were found to develop simultaneously with the increase in diameter. Energy-dispersed X-ray analysis and back-scattered electron microscopy showed that the whitish clusters contained mainly organic matter and no inorganic calcium precipitates. The white clusters had a higher density than the black matrix, as evidenced by the denser cell arrangement observed by high-magnification electron microscopy and the significantly higher effective diffusion coefficient determined by nuclear magnetic resonance imaging. High-magnification electron microscopy indicated a segregation of acetate-utilizing methanogens (Methanosaeta spp.) in the white clusters from syntrophic species and hydrogenotrophic methanogens (Methanobacterium-like and Methanospirillum-like organisms) in the black matrix. A number of physical and microbial ecology reasons for the observed structure are proposed, including the advantage of segregation for high-rate degradation of syntrophic substrates.     

Microbial diversity and activity through a permafrost/ground ice core profile from the Canadian high Arctic

Culture‐dependent and culture‐independent methods were used in an investigation of the microbial diversity in a permafrost/massive ground ice core from the Canadian high Arctic. Denaturing gradient gel electrophoresis as well as Bacteria and Archaea 16S rRNA gene clone libraries showed differences in the composition of the microbial communities in the distinct core horizons. Microbial diversity was similar in the active layer (surface) soil, permafrost table and permafrost horizons while the ground ice microbial community showed low diversity. Bacteria and Archaea sequences related to the Actinobacteria (54%) and Crenarchaeota (100%) respectively were predominant in the active layer while the majority of sequences in the permafrost were related to the Proteobacteria (57%) and Euryarchaeota (76%). The most abundant phyla in the ground ice clone libraries were the Firmicutes (59%) and Crenarchaeota (82%). Isolates from the permafrost were both less abundant and diverse than in the active layer soil, while no culturable cells were recovered from the ground ice. Mineralization of [1‐¹⁴C] acetic acid and [2‐¹⁴C] glucose was used to detect microbial activity in the different horizons in the core. Mineralization was detected at near ambient permafrost temperatures (?15°C), indicating that permafrost may harbour an active microbial population, while the low microbial diversity, abundance and activity in ground ice suggests a less hospitable microbial habitat.     

Confocal imaging of in situ natural microbial communities and their extracellular polymeric secretions using Nanoplast resin

A novel method using excision and fixation in Nanoplast, a hydrophilic embedding resin, allows confocal imaging of natural microbial communities and their extracellular polymeric secretions (EPS) while in situ. Prestaining with fluorescent probes permits the observation of specific cellular and extracellular components. Marine stromatolite sediments were examined using this method. Optical sectioning using confocal laser scanning microscopy (CLSM) permitted high-resolution imaging through sediments. Delicate arrangements of the EPS that are associated with sedimentary microbial biofilms were imaged using a fluorescein isothiocyanate (FITC)-labeled lectin (concanavalin-A) probe. Close microspatial associations of heterotrophic bacteria cells and autotrophic cyanobacteria cells were also observed. The nanoplast resin produces no detectable autofluorescence. Further coupling of multi-photon scanning laser microscopy (2P-LSM) with a conventional single photon CLSM allowed concurrent imaging of DAPI-labeled microbial cells, FITC-labeled EPS and autofluorescent carbonate sand grains. The multi-photon infrared laser permits deep (approximately 1 mm) penetration of samples and the excitation of DAPI, which normally requires UV-excitation with minimal disturbance to samples. The unique combination of Nanoplast with fluorescent probes, CLSM and 2P-LSM allows for the preservation and imaging of natural microbial communities in their in situ state, a method easily adapted for examinations of other microbial systems.     

Fluorescence in situ hybridisation coupled to ultra small immunogold detection to identify prokaryotic cells using transmission and scanning electron microscopy

We describe a method based on fluorescence in situ hybridisation (FISH) that allows the identification of individual cells by electron microscopy. We hybridised universal and specific fluorescein-labelled oligonucleotide probes to the ribosomal RNA of prokaryotic microorganisms in heterogeneous cell mixtures. We then used antibodies against fluorescein coupled to sub-nanometer gold particles to label the hybridised probes in the ribosome. After increasing the diameter of the metal particles by silver enhancement, the specific gold-silver signal was visualised by optical microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is the first time that SEM is applied to the detection of gold nanoparticles hybridised to an intracellular target, such as the ribosome. The possibility to couple phylogenetic identification by FISH to cell surface and ultrastructure observation at electron microscopy resolution has promising potential applications in microbial ecology.     

Ultrastructural changes of platelets during ADP- stimulated aggregation (in the presence of fibrinogen)

The ultrastructure of resting and stimulated human blood platelets (P) was studied by the transmission electron microscopy. The cells were chemically fixed (using tannic acid and OsFeCN mixture) 1, 3, 5 and 15 min after the addition of ADP and fibrinogen (F). Early changes in P ultrastructure consist in drastic reduction of the electron-dense layer of glycocalyx and in an increase of the plasma membrane permeability. At the early stages of P aggregation the cells contact with each other due to rapidly arising pseudopodia. Later, the extracellular network containing an exogenous F participates in the aggregation process.     

Electricity Generation in Microbial Fuel Cells Using Neutral Red as an Electronophore

Neutral red (NR) was utilized as an electron mediator in microbial fuel cells consuming glucose to study both its efficiency during electricity generation and its role in altering anaerobic growth and metabolism of Escherichia coli and Actinobacillus succinogenes. A study of chemical fuel cells in which NADH, NR, and ferricyanide were the electron donor, the electronophore, and the electron acceptor, respectively, showed that electrical current produced from NADH was proportional to the concentration of NADH. Fourfold more current was produced from NADH in chemical fuel cells when NR was the electron mediator than when thionin was the electron mediator. In microbial fuel cells in which E. coli resting cells were used the amount of current produced from glucose when NR was the electron mediator (3.5 mA) was 10-fold more than the amount produced when thionin was the electron mediator (0.4 mA). The amount of electrical energy generated (expressed in joules per mole of substrate) and the amount of current produced from glucose (expressed in milliamperes) in NR-mediated microbial fuel cells containing either E. coli or A. succinogenes were about 10- and 2-fold greater, respectively, when resting cells were used than when growing cells were used. Cell growth was inhibited substantially when these microbial fuel cells were making current, and more oxidized end products were formed under these conditions. When sewage sludge (i.e., a mixed culture of anaerobic bacteria) was used in the fuel cell, stable (for 120 h) and equivalent levels of current were obtained with glucose, as observed in the pure-culture experiments. These results suggest that NR is better than other electron mediators used in microbial fuel cells and that sludge production can be decreased while electricity is produced in fuel cells. Our results are discussed in relation to factors that may improve the relatively low electrical efficiencies (1.2 kJ/mol) obtained with microbial fuel cells.     

Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy

Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of microscale associations. Electron microscopy has been used extensively for geomicrobial investigations, and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions by conventional electron microscopy approaches with imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding the nature of interactions between microbial extracellular polymers and their environment.     

Immunological Detection of Nitrospira-like Bacteria in Various Soils

Chemolithotrophic nitrite oxidizers were enriched from five different soils including freshwater marsh, permafrost, garden, agricultural, and desert soils and monitored during the cultivation procedure. Immunoblot analysis was used to identify the nitrite oxidizing organisms with monoclonal antibodies, which recognize the key enzyme of nitrite oxidation in a genus-specific reaction [Bartosch et al. (1999) Appl Environ Microbiol 65:4126-4133]. The morphological characteristics of the enriched nitrite oxidizers were additionally studied using transmission electron microscopy (TEM) and fluorescence microscopy. By means of the antibodies and TEM analysis Nitrospira could be clearly identified in enrichment cultures derived from freshwater marsh and from permafrost soil. Nitrospira cells were enriched simultaneously with cells of the genus Nitrobacter when nitrite concentrations of 0.2 g of NaNO2 L(-1) were used. However, in enrichment cultures containing 2 g of NaNO2 L(-1) Nitrobacter was exclusively detected. During fluorescence microscopic observations of DAPI stained samples microcolonies were found in enrichment cultures from freshwater marsh, permafrost, garden, and agricultural soil. They had a similar morphology to Nitrospira-like microcolonies from activated sludge. In conclusion, Nitrospira seems to be not only a common aquatic but also a usual soil bacterium.     

Microbial communities of ancient seeds derived from permanently frozen Pleistocene deposits

Microbial communities from the surface of ancient seeds of higher plants and embedding frozen material dated to the late Pleistocene (formed about 30 thousand years ago) were studied by various methods: scanning electron microscopy, epifluorescence microscopy, and inoculation of nutrient media, followed by identification of isolated cultures. Both prokaryotic and eukaryotic microorganisms were found on the surface of ancient seeds. The total quantity of bacterial cells determined by direct counting and dilution plating (CFU) for the samples of ancient seeds exceeded the value in the embedding frozen material by one to two orders of magnitude. This pattern was not maintained for mycelial fungi; their quantity in the embedding material was also rather high. A significant difference was revealed between the microbial communities of ancient seeds and embedding frozen material. These findings suggest that ancient plant seeds are a particular ecological niche for microorganisms existing in permafrost and require individual detailed study.